MULTITARGET DRUGS: VERIFICATION-OF-PRINCIPLE STUDIES

药物选择性不足是当今疗法的一大祸害。这一问题对抗菌药物来说是严重的，对抗病毒药物来说是困难的，对抗癌药物来说是完全没有解决的，因为抗癌药物对主要癌症仍然无效，而且还会产生严重的副作用。如果治疗剂能够获得多靶点、组合选择性、杀死(或以其他方式修饰)一个细胞，当且仅当它包含一组预定的分子靶点而缺乏另一组预定的靶点时，这个问题就可能得到解决。最近，有人提出了一种适用于小分子(小于或等于1kD)化合物的药物选择性问题的解决方案(Varshavsky，A.PNAS 95,2094-2099(1998))。干扰/共显性(IC)和反效应子(IC)这两个概念应该允许治疗方案具有组合选择性，其中正负向感应的大分子靶点的数量可以是两个、三个或更多。这类药物在体内的作用类似于布尔运算“与”、“非”或“非”及其组合的结果。建议的研究将得到本R21申请的支持，将验证以酿酒酵母为模式生物的多目标设计的原则。具体目标1)泛素融合技术将被用于在酵母中表达和分析一组在体内环境中作为IC型化合物的多部分线性多肽。2)将合成包括无氢四环素(AT)部分和与AT连接的甲氨蝶呤(MTX)或三甲氧氨蝶呤(TM)部分的两部分IC型化合物，然后在体外(与纯化的TetR或DHFR结合的互斥性)和在体内(对缺乏或表达AT结合的E.ColiTetR阻遏物的酿酒酵母的不同毒性)进行表征。3)合成含有新诺维菌素(Nov)和甲氨蝶呤(MTX)或甲氨蝶呤(Tm)部分的两部分IC型化合物，并测试其与缺乏或表达Nov结合的E.coliDNA GyrB亚单位的酿酒酵母的差异细胞毒性。4)合成含有At部分、β-蜕皮激素(EC)部分、甲氨蝶呤(MTX)或甲氨蝶呤(Tm)部分的三元IC型化合物，并与缺乏或表达相应大分子靶标的酿酒酵母及其配对组合进行差异毒性测试。

The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anti-cancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. T he problem may be solved if therapeutic agents could acquire a multi-target, combinatorial selectivity, killing (or otherwise modifying) a cell if, and only if, it contains a predetermined set of molecular targets and lacks another predetermined set of targets. Recently, a solution to the problem of drug selectivity was proposed that is applicable to small (less than or equal to 1 kD) compounds (Varshavsky, A. PNAS 95, 2094-2099 (1998)). Two ideas, interference/co-dominance (IC) and anti-effectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations "and", "or" "not", and combinations thereof. The proposed studies, to be supported by the present R21 application, will verify the principles of multi-target designs using the yeast Saccharomyces cerevisiae as a model organism. Specific Aims 1) The ubiquitin fusion technique will be used to express and analyze, in the yeast S. cerevisiae, a set of multi-moiety linear peptides that act as IC- type compounds in this in vivo setting. 2) Bipartite, IC-type compounds that comprise the anhydrotetracycline (at) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety linked to at will be synthesized, and thereafter characterized in vitro (mutual exclusivity of binding to the purified TetR or DHFR) and in vivo (differential toxicity to S. cerevisiae that either lack or express the at- binding E. coli TetR repressor). 3) Bipartite, IC-type compounds containing the novobiocin (nov) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential cytotoxicity with S. cerevisiae that either lack or express the nov-binding B-subunit of E. coli DNA gyrates (GyrB). 4) Tripartite, IC-type compounds containing the at moiety, the beta- ecdysone (ec) moiety, and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential toxicity with S. cerevisiae that lack or express the corresponding macromolecular targets and their pairwise combinations.